Membrane Heteroreceptor Complexes as Second-Order Protein Modulators: A Novel Integrative Mechanism through Allosteric Receptor-Receptor Interactions.

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR-D2R, GABAA-D5R, and FGFR1-5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor-receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor-receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor-receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000-2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor-receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor-receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson's disease.

Combined treatment with Sigma1R and A2AR agonists fails to inhibit cocaine self-administration despite causing strong antagonistic accumbal A2AR-D2R complex interactions: the potential role of astrocytes.

Previous studies have indicated that acute treatment with the monoamine stabilizer OSU-6162 (5 mg/kg), which has a high affinity for Sigma1R, significantly increased the density of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes following cocaine self-administration. Ex vivo studies using the A2AR agonist CGS21680 also suggested the existence of enhanced antagonistic accumbal A2AR-D2R allosteric interactions after treatment with OSU-6162 during cocaine self-administration. However, a 3-day treatment with OSU-6162 (5 mg/kg) failed to alter the behavioral effects of cocaine self-administration. To test these results and the relevance of OSU-6162 (2.5 mg/kg) and/or A2AR (0.05 mg/kg) agonist interactions, we administered low doses of receptor agonists during cocaine self-administration and assessed their neurochemical and behavioral effects. No effects were observed on cocaine self-administration; however, marked and highly significant increases using the proximity ligation assay (PLA) were induced by the co-treatment on the density of the A2AR-D2R heterocomplexes in the nucleus accumbens shell. Significant decreases in the affinity of the D2R high- and low-affinity agonist binding sites were also observed. Thus, in low doses, the highly significant neurochemical effects observed upon cotreatment with an A2AR agonist and a Sigma1R ligand on the A2AR-D2R heterocomplexes and their enhancement of allosteric inhibition of D2R high-affinity binding are not linked to the modulation of cocaine self-administration. The explanation may be related to an increased release of ATP and adenosine from astrocytes in the nucleus accumbens shell in cocaine self-administration. This can lead to increased activation of the A1R protomer in a putative A1R-A2AR-D2R complex that modulates glutamate release in the presynaptic glutamate synapse. We hypothesized that the integration of changes in presynaptic glutamate release and postjunctional heteroreceptor complex signaling, where D2R plays a key role, result in no changes in the firing of the GABA anti-reward neurons, resulting in no reduction in cocaine self-administration in the present experiments.

The mGlu5 Receptor Protomer-Mediated Dopamine D2 Receptor Trans-Inhibition Is Dependent on the Adenosine A2A Receptor Protomer: Implications for Parkinson's Disease.

The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.

Increased density and antagonistic allosteric interactions in A2AR-D2R heterocomplexes in extinction from cocaine use, lost in cue induced reinstatement of cocaine seeking.

Neurochemical studies were previously performed on the effects of a 10 day extinction learning from cocaine self-administration on D2R and A2AR recognition and D2R Gi/o coupling in the ventral striatum. In the present study biochemical receptor binding and proximity ligation assay were used to study possible changes in the allosteric receptor-receptor interactions and the density of the A2AR-D2R heterocomplexes in the ventral striatum (nucleus accumbens shell) in extinction from cocaine self-administration including cue induced reinstatement of cocaine seeking. A significant and clear-cut reduction of active lever pressing was observed in extinction on day 10 from cocaine use. In cue induced reinstatement of cocaine self-administration a significant return in active lever presses developed. In extinction, significant increases in the density of A2AR-D2R and D2R-Sigma1R heterocomplexes were observed in nucleus accumbens shell. In contrast, cue-induced reinstatement of cocaine seeking produced no significant changes in these heteroreceptor complexes of the nucleus accumbens shell. In the 3H raclopride/quinpirole competition binding experiments, the extinction led to a significant increase in the D2R Ki, High dissociation constant in the ventral striatum upon ex vivo exposure to CGS 21680 (100 nM), compared to the same exposure performed in membrane preparations from yoked saline rats. No significant changes in D2R Ki, High values were observed in membrane preparations from rats after cue-induced reinstatement of cocaine-seeking undergoing the same exposure ex vivo to CGS 21680 when compared with membrane preparations from yoked saline rats undergoing the same procedures. It seems likely that increased formation of A2AR-D2R and putative A2AR-D2R-Sigma1R heterocomplexes in the nucleus accumbens shell is part of the mechanism for the enhanced antagonistic allosteric A2AR-D2R interactions developed in extinction learning from cocaine. It reduces cocaine reward through reduced D2R function, and these inhibitory mechanisms are no longer in operation in cue induced reinstatement of cocaine seeking.

The coming together of allosteric and phosphorylation mechanisms in the molecular integration of A2A heteroreceptor complexes in the dorsal and ventral striatal-pallidal GABA neurons.

The role of adenosine A2A receptor (A2AR) and striatal-enriched protein tyrosine phosphatase (STEP) interactions in the striatal-pallidal GABA neurons was recently discussed in relation to A2AR overexpression and cocaine-induced increases of brain adenosine levels. As to phosphorylation, combined activation of A2AR and metabotropic glutamate receptor 5 (mGluR5) in the striatal-pallidal GABA neurons appears necessary for phosphorylation of the GluA1 unit of the AMPA receptor to take place. Robert Yasuda (J Neurochem 152: 270-272, 2020) focused on finding a general mechanism by which STEP activation is enhanced by increased A2AR transmission in striatal-pallidal GABA neurons expressing A2AR and dopamine D2 receptor. In his Editorial, he summarized in a clear way the significant effects of A2AR activation on STEP in the dorsal striatal-pallidal GABA neurons which involves a rise of intracellular levels of calcium causing STEP activation through its dephosphorylation. However, the presence of the A2AR in an A2AR-fibroblast growth factor receptor 1 (FGFR1) heteroreceptor complex can be required in the dorsal striatal-pallidal GABA neurons for the STEP activation. Furthermore, Won et al. (Proc Natl Acad Sci USA 116: 8028-8037, 2019) found in mass spectrometry experiments that the STEP splice variant STEP61 can bind to mGluR5 and inactivate it. In addition, A2AR overexpression can lead to increased formation of A2AR-mGluR5 heterocomplexes in ventral striatal-pallidal GABA neurons. It involves enhanced facilitatory allosteric interactions leading to increased Gq-mediated mGluR5 signaling activating STEP. The involvement of both A2AR and STEP in the actions of cocaine on synaptic downregulation was also demonstrated. The enhancement of mGluR5 protomer activity by the A2AR protomer in A2AR-mGluR5 heterocomplexes in the nucleus accumbens shell appears to have a novel significant role in STEP mechanisms by both enhancing the activation of STEP and being a target for STEP61.

The Balance of MU-Opioid, Dopamine D2 and Adenosine A2A Heteroreceptor Complexes in the Ventral Striatal-Pallidal GABA Antireward Neurons May Have a Significant Role in Morphine and Cocaine Use Disorders.

The widespread distribution of heteroreceptor complexes with allosteric receptor-receptor interactions in the CNS represents a novel integrative molecular mechanism in the plasma membrane of neurons and glial cells. It was proposed that they form the molecular basis for learning and short-and long-term memories. This is also true for drug memories formed during the development of substance use disorders like morphine and cocaine use disorders. In cocaine use disorder it was found that irreversible A2AR-D2R complexes with an allosteric brake on D2R recognition and signaling are formed in increased densities in the ventral enkephalin positive striatal-pallidal GABA antireward neurons. In this perspective article we discuss and propose how an increase in opioid heteroreceptor complexes, containing MOR-DOR, MOR-MOR and MOR-D2R, and their balance with each other and A2AR-D2R complexes in the striatal-pallidal enkephalin positive GABA antireward neurons, may represent markers for development of morphine use disorders. We suggest that increased formation of MOR-DOR complexes takes place in the striatal-pallidal enkephalin positive GABA antireward neurons after chronic morphine treatment in part through recruitment of MOR from the MOR-D2R complexes due to the possibility that MOR upon morphine treatment can develop a higher affinity for DOR. As a result, increased numbers of D2R monomers/homomers in these neurons become free to interact with the A2A receptors found in high densities within such neurons. Increased numbers of A2AR-D2R heteroreceptor complexes are formed and contribute to enhanced firing of these antireward neurons due to loss of inhibitory D2R protomer signaling which finally leads to the development of morphine use disorder. Development of cocaine use disorder may instead be reduced through enkephalin induced activation of the MOR-DOR complex inhibiting the activity of the enkephalin positive GABA antireward neurons. Altogether, we propose that these altered complexes could be pharmacological targets to modulate the reward and the development of substance use disorders.

Molecular, biochemical and behavioural evidence for a novel oxytocin receptor and serotonin 2C receptor heterocomplex.

The complexity of oxytocin-mediated functions is strongly associated with its modulatory effects on other neurotransmission systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Signalling between oxytocin (OT) and 5-HT has been demonstrated during neurodevelopment and in the regulation of specific emotion-based behaviours. It is suggested that crosstalk between neurotransmitters is driven by interaction between their specific receptors, particularly the oxytocin receptor (OTR) and the 5-hydroxytryptamine 2C receptor (5-HTR2C), but evidence for this and the downstream signalling consequences that follow are lacking. Considering the overlapping central expression profiles and shared involvement of OTR and 5-HTR2C in certain endocrine functions and behaviours, including eating behaviour, social interaction and locomotor activity, we investigated the existence of functionally active OTR/5-HTR2C heterocomplexes. Here, we demonstrate evidence for a potential physical interaction between OTR and 5-HTR2Cin vitro in a cellular expression system using flow cytometry-based FRET (fcFRET). We could recapitulate this finding under endogenous expression levels of both receptors via in silico analysis of single cell transcriptomic data and ex vivo proximity ligation assay (PLA). Next, we show that co-expression of the OTR/5-HTR2C pair resulted in a significant depletion of OTR-mediated Gαq-signalling and significant changes in receptor trafficking. Of note, attenuation of OTR-mediated downstream signalling was restored following pharmacological blockade of the 5-HTR2C. Finally, we demonstrated a functional relevance of this novel heterocomplex, in vivo, as 5-HTR2C antagonism increased OT-mediated hypoactivity in mice. Overall, we provide compelling evidence for the formation of functionally active OTR/5-HTR2C heterocomplexes, adding another level of complexity to OTR and 5-HTR2C signalling functionality. This article is part of the special issue on Neuropeptides.

Adenosine A2AReceptors in Substance Use Disorders: A Focus on Cocaine.

Several psychoactive drugs can evoke substance use disorders (SUD) in humans and animals, and these include psychostimulants, opioids, cannabinoids (CB), nicotine, and alcohol. The etiology, mechanistic processes, and the therapeutic options to deal with SUD are not well understood. The common feature of all abused drugs is that they increase dopamine (DA) neurotransmission within the mesocorticolimbic circuitry of the brain followed by the activation of DA receptors. D2 receptors were proposed as important molecular targets for SUD. The findings showed that D2 receptors formed heteromeric complexes with other GPCRs, which forced the addiction research area in new directions. In this review, we updated the view on the brain D2 receptor complexes with adenosine (A)2A receptors (A2AR) and discussed the role of A2AR in different aspects of addiction phenotypes in laboratory animal procedures that permit the highly complex syndrome of human drug addiction. We presented the current knowledge on the neurochemical in vivo and ex vivo mechanisms related to cocaine use disorder (CUD) and discussed future research directions for A2AR heteromeric complexes in SUD.

Evidence for the existence of A2AR-TrkB heteroreceptor complexes in the dorsal hippocampus of the rat brain: Potential implications of A2AR and TrkB interplay upon ageing.

Adenosine A2A receptors (A2AR) are crucial in facilitating the BDNF action on synaptic transmission in the rat hippocampus primarily upon ageing. Furthermore, it has been suggested that A2AR-Tropomyosin related kinase B receptor (TrkB) crosstalk has a pivotal role in adenosine A2AR-mediated modulation of the BDNF action on hippocampal plasticity. Considering the impact of the above receptors interplay on what concerns BDNF-induced enhancement of synaptic transmission, gaining a better insight into the mechanisms behind this powerful crosstalk becomes of primary interest. Using in situ proximity ligation assay (PLA), the existence of a direct physical interaction between adenosine A2AR and TrkB is demonstrated. The A2AR-TrkB heteroreceptor complexes show a heterogeneous distribution within the rat dorsal hippocampus. High densities of the heteroreceptor complexes were observed in the pyramidal cell layers of CA1-CA3 regions and in the polymorphic layer of the dentate gyrus (DG). The stratum radiatum of the CA1-3 regions showed positive PLA signal in contrast to the oriens region. The molecular and granular layers of the DG also lacked significant densities of PLA positive heteroreceptor complexes, but subgranular zone showed some PLA positive cells. Their allosteric receptor-receptor interactions may significantly modulate BDNF signaling impacting on hippocampal plasticity which is impaired upon ageing.

Multiple Adenosine-Dopamine (A2A-D2 Like) Heteroreceptor Complexes in the Brain and Their Role in Schizophrenia.

In the 1980s and 1990s, the concept was introduced that molecular integration in the Central Nervous System could develop through allosteric receptor-receptor interactions in heteroreceptor complexes presents in neurons. A number of adenosine-dopamine heteroreceptor complexes were identified that lead to the A2A-D2 heteromer hypothesis of schizophrenia. The hypothesis is based on strong antagonistic A2A-D2 receptor-receptor interactions and their presence in the ventral striato-pallidal GABA anti-reward neurons leading to reduction of positive symptoms. Other types of adenosine A2A heteroreceptor complexes are also discussed in relation to this disease, such as A2A-D3 and A2A-D4 heteroreceptor complexes as well as higher order A2A-D2-mGluR5 and A2A-D2-Sigma1R heteroreceptor complexes. The A2A receptor protomer can likely modulate the function of the D4 receptors of relevance for understanding cognitive dysfunction in schizophrenia. A2A-D2-mGluR5 complex is of interest since upon A2A/mGluR5 coactivation they appear to synergize in producing strong inhibition of the D2 receptor protomer. For understanding the future of the schizophrenia treatment, the vulnerability of the current A2A-D2like receptor complexes will be tested in animal models of schizophrenia. A2A-D2-Simag1R complexes hold the highest promise through Sigma1R enhancement of inhibition of D2R function. In line with this work, Lara proposed a highly relevant role of adenosine for neurobiology of schizophrenia.

Acute cocaine treatment enhances the antagonistic allosteric adenosine A2A-dopamine D2 receptor-receptor interactions in rat dorsal striatum without increasing significantly extracellular dopamine levels.

BACKGROUND: Antagonistic adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) receptor-receptor interactions have previously been demonstrated in A2AR-D2R heteroreceptor complexes in the rat dorsal striatum. They mainly involve a reduction of affinity in the high-affinity component of the D2R agonist binding site upon activation in vivo of the A2AR by an A2AR agonist. Upon cocaine self-administration, this antagonistic A2AR-D2R interaction disappeared in the dorsal striatum. METHODS: In the current experiments, it was tested whether such modifications in the antagonistic A2AR-D2R receptor-receptor interactions can develop also after an acute systemic injection of a low cocaine dose (1 mg/kg; sc). RESULTS: Microdialysis experiments indicated that acute cocaine did not significantly alter the extracellular dopamine levels in the dorsal striatum of the awake Wistar rats. Competition dopamine receptor binding experiments demonstrated that in the acute cocaine group, the A2AR agonist CGS-21680 produced significantly larger increases in the D2R Ki, High values (reduction of high-affinity) versus the saline-injected (i.e. control) group. Furthermore, in the dorsal striatum membrane preparation from acute cocaine-injected rats, CGS-21680 also produced significant increases in the D2R Ki, Low values (reduction of low-affinity) and in the proportion of D2Rs in the high-affinity state (RH). Such significant effects were not observed with CGS-21680 in the control group. CONCLUSIONS: The molecular mechanism involved in the acute cocaine-induced increase in the antagonistic allosteric A2AR-D2R receptor-receptor interactions may be an increased formation of higher-order complexes A2AR-D2R-sigma1R in which cocaine by binding to the sigma1R protomer also allosterically enhances the inhibitory A2AR-D2R interaction in this receptor complex.

A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but Not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration.

It was previously demonstrated that rat adenosine A2AR transmembrane V peptide administration into the nucleus accumbens enhances cocaine self-administration through disruption of the A2AR-dopamine (D2R) heteroreceptor complex of this region. Unlike human A2AR transmembrane 4 (TM4) and 5 (TM5), A2AR TM2 did not interfere with the formation of the A2AR-D2R heteroreceptor complex in cellular models using BRET1 assay. A2AR TM2 was proposed to be part of the of the receptor interface of the A2AR homomer instead and was therefore tested in the current article for effects on rat cocaine self-administration using rat A2AR synthetic TM2 peptide bilaterally injected into the nucleus accumbens. The injected A2AR TM2 peptide failed to significantly counteract the inhibitory action of the A2AR agonist CGS 21680 (0.1 mg/Kg) on cocaine self-administration. In line with these results, the microinjected A2AR TM2 peptide did not reduce the number of proximity ligation assay blobs identifying A2AR-D2R heteroreceptor complexes in the nucleus accumbens. In contrast, the A2AR TM2 peptide significantly reduced the number of A2AR-A2AR homoreceptor complexes in the nucleus accumbens. As to effects on the receptor-receptor interactions in the A2AR-D2R heteroreceptor complexes, the A2AR TM2 peptide did not alter the significant increase in the D2R Ki, high values produced by the A2AR agonist CGS 21680 ex vivo in the ventral striatum. The results indicate that the accumbal A2AR-A2AR homomeric complexes are not involved in mediating the A2AR agonist-induced inhibition of cocaine self-administration.

Can Allosteric Receptor-Protein Interactions in Receptor Complexes Be a Molecular Mechanism Involved in Cancer Immune Therapy?

Based on the work in the Central Nervous System with discoveries of allosteric receptor-receptor interactions in homo- and heteroreceptor complexes representing a major integrative mechanism in synapses and extrasynaptic regions, it is proposed that a similar mechanism may exist in the immunological synapses. We discuss a putative additional molecular mechanism for the ability of the inhibitory T cell signaling proteins CTLA-4 and PD-1 and the adenosine A2AR to diminish T cell activation leading to enhancement of cancer development. We suggest that in the same immunological synapse involving T cells and antigen presenting cells multiple heteroreceptor complexes may participate and be in balance with each other. Their composition can vary between functional states and among different types of T cells. The T cell receptor (TCR) and its accelerators, strongly enhancing T cell activation, can be under inhibitory control by T cell signaling proteins CTLA4 and PD-1 and also the adenosine A2AR through inhibitory allosteric receptor-receptor interactions in different types of heteroreceptor complexes. As a result, inhibitory tumor induced immunosuppression can develop due to a dominance of the inhibitory signaling causing a brake on the TCR and/or its accelerator and the cancer immunotherapy becomes blocked.

Potentiation of cannabinoid signaling in microglia by adenosine A2A receptor antagonists.

Neuroprotective M2-skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein-coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A2A (A2A R) and cannabinoid CB2 (CB2 R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2 R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2A R antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A -CB2 receptor heteromer (A2A -CB2 Het). Particularly relevant is the upregulation of A2A -CB2 Het expression in samples from the APPSw ,Ind AD transgenic mice model. The most relevant finding, confirmed in both heterologous cells and in primary cultures of microglia, was that blockade of A2A receptors results in increased CB2 R-mediated signaling. This heteromer-specific feature suggests that A2A R antagonists would potentiate, via microglia, the neuroprotective action of endocannabinoids with implications for AD therapy.

Adenosine heteroreceptor complexes in the basal ganglia are implicated in Parkinson's disease and its treatment.

The adenosine homo, iso and heteroreceptor complexes in the basal ganglia play a highly significant role in modulating the indirect and direct pathways and the striosomal projections to the nigro-striatal DA system. The major adenosine receptor complexes in the striato-pallidal GABA neurons can be the A2AR-D2R and A2AR-D2R-mGluR5 receptor complexes, in which A2AR protomers and mGluR5 protomers can allosterically interact to inhibit D2R protomer signaling. Through a reorganization of these heteroreceptor complexes upon chronic dopaminergic treatment a pathological and prolonged inhibition of D2R receptor protomer signaling can develop with motor inhibition and wearing off of the therapeutic effects of levodopa and dopamine receptor agonists. The direct pathway is enriched in D1R in and around glutamate synapses enhancing the ability of these GABA neurons to be activated and increase motor initiation. The brake on these GABA neurons is in this case exerted by A1R forming A1R-D1R heteroreceptor complexes in which they allosterically inhibit D1R signaling and thereby reduce motor initiation. Upon chronic levodopa treatment a reorganization of the D1R heteroreceptor complexes develops with the formation of putative A1R-D1R-D3 in addition to D1R-D3R complexes in which D3R enhances D1R protomer signaling and may make the A1R protomer brake less effective. Alpha-synuclein monomers-dimers are postulated to form complexes with A2AR homo and heteroprotomers in the plasma membrane enhancing alpha-synuclein aggregation and toxicity. The alpha-synuclein fibrils formed in the A2AR enriched dendritic spines of the striato-pallidal GABA neurons may reach the surrounding DA terminals via extracellular-vesicle-mediated volume transmission involving internalization of the vesicles and their cargo (alpha-synuclein fibrils) into the vulnerable DA terminals, enhancing their degeneration followed by retrograde flow of these fibrils in the DA axons to the vulnerable nigral DA nerve cells.

A2AR-D2R Heteroreceptor Complexes in Cocaine Reward and Addiction.

The concept of allosteric receptor-receptor interactions in G protein-coupled receptor homo- and heteroreceptor complexes in which they physically interact provides a new dimension to molecular integration in the brain. The receptor-receptor interactions dynamically change recognition, pharmacology, signaling, and trafficking of the participating receptors. Among the receptor complexes, disruption of the A2A receptor-dopamine D2 receptor (A2AR-D2R) complex by an A2AR agonist has been shown to fully block the inhibition of cocaine self-administration. Cocaine induced pathological A2AR-D2R-Sigma1R complexes may form a long-term memory with a strong and permanent D2R brake, leading to cocaine addiction. These heteroreceptor complexes can potentially be targeted for future pharmacotherapy of cocaine addiction by using heterobivalent compounds or A2AR-D2R receptor interface-interfering peptides that disrupt the A2AR-D2R-Sigma1R complexes.

Mapping the Interface of a GPCR Dimer: A Structural Model of the A2A Adenosine and D2 Dopamine Receptor Heteromer.

The A2A adenosine (A2AR) and D2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A2AR-D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A2AR-D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A2AR-D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A2AR-D2R receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A2AR blocked heterodimer interactions and disrupted the allosteric effect of A2AR activation on D2R agonist binding. Protein-protein docking was used to construct a model of the A2AR-D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A2AR-D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A2AR-D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

Dopamine D2 Receptor Supersensitivity as a Spectrum of Neurotoxicity and Status in Psychiatric Disorders.

Abnormality of dopamine D2 receptor (D2R) function, often observed as D2R supersensitivity (D2RSS), is a commonality of schizophrenia and related psychiatric disorders in humans. Moreover, virtually all psychotherapeutic agents for schizophrenia target D2R in brain. Permanent D2RSS as a feature of a new animal model of schizophrenia was first reported in 1991, and then behaviorally and biochemically characterized over the next 15-20 years. In this model of schizophrenia characterized by production of D2RSS in ontogeny, there are demonstrated alterations of signaling processes, as well as functional links between the biologic template of the animal model and ability of pharmacotherapeutics to modulate or reverse biologic and behavioral modalities toward normality. Another such animal model, featuring knockout of trace amine-associated receptor 1 (TAAR1), demonstrates D2RSS with an increase in the proportion of D2R in the high-affinity state. Currently, TAAR1 agonists are being explored as a therapeutic option for schizophrenia. There is likewise an overlay of D2RSS with substance use disorder. The aspect of adenosine A2A-D2 heteroreceptor complexes in substance use disorder is highlighted, and the association of adenosine A2A receptor antagonists in discriminative and rewarding effects of psychostimulants is outlined. In summary, these new animal models of schizophrenia have face, construct, and predictive validity, and distinct advantages over earlier models. While the review summarizes elements of D2RSS in schizophrenia per se, and its interplay with substance use disorder, a major focus is on presumed new molecular targets attending D2RSS in schizophrenia and related clinical entities.

Understanding the Role of Adenosine A2AR Heteroreceptor Complexes in Neurodegeneration and Neuroinflammation.

Adenosine is a nucleoside mainly formed by degradation of ATP, located intracellularly or extracellularly, and acts as a neuromodulator. It operates as a volume transmission signal through diffusion and flow in the extracellular space to modulate the activity of both glial cells and neurons. The effects of adenosine are mediated via four adenosine receptor subtypes: A1R, A2AR, A2BR, A3R. The A2AR has a wide-spread distribution but it is especially enriched in the ventral and dorsal striatum where it is mainly located in the striato-pallidal GABA neurons at a synaptic and extrasynaptic location. A number of A2AR heteroreceptor complexes exist in the striatum. The existence of A2AR-D2R heteroreceptor complexes with antagonistic A2AR-D2R interactions in the striato-pallidal GABA neurons is well-known with A2AR activation inhibiting Gi/o mediated signaling of D2Rs. A2AR-mGluR5 heteroreceptor complexes were also found in with synergistic receptor-receptor interactions enhancing the inhibition of the D2R protomer signaling. They are located mainly in extrasynaptic regions of the striato-pallidal GABA neurons. Results recently demonstrated the existence of brain A2AR-A2BR heteroreceptor complexes, in which A2BR protomer constitutively inhibited the function of the A2AR protomer. These adenosine A2AR heteroreceptor complexes may modulate alpha-synuclein aggregation and toxicity through postulated bidirectional direct interactions leading to marked increases in A2AR signaling both in nerve cells and microglia. It is of high interest that formation of A2AR-A2ABR heteroreceptor complexes provides a brake on A2AR recognition and signaling opening up a novel strategy for treatment of A2AR mediated neurodegeneration.